PROJECT SUMMARY Protein labeling by fusion with genetically encoded fluorescent proteins has been a powerful tool for investigating biological processes, allowing scientists to observe and analyze protein expression, localization, and dynamics in living cells. However, traditional approaches for expressing fluorescent fusion proteins possess drawbacks including potential overexpression artifacts, and new methods are needed, especially for in vivo studies. Our lab studies vertebrate organ formation using the zebrafish (Danio rerio) model system. In zebrafish and many other model organisms, expression of fluorescent fusion proteins is often achieved by injection of in vitro transcribed mRNA, which provides ubiquitous expression, or by transgenesis, which utilizes gene regulatory elements to drive spatially and/or temporally restricted expression. However, both approaches run the risk of producing overexpression artifacts. An alternative approach is to ?knock-in? fluorescent coding sequences into the genetic locus for the protein of interest. Although this approach preserves endogenous regulation of expression, targeted insertion can be technically difficult to achieve. Moreover, many proteins are expressed quite broadly, and fluorescent protein tagging at the endogenous locus does not allow one to study the tissue- specific roles of such proteins. To overcome these limitations, we propose using a split fluorescent protein approach to achieve tissue-specific and endogenous protein labeling. Split fluorescent proteins consist of protein fragments that are expressed independently and possess little to no fluorescence on their own. However, when present in the same cell, the fragments self-assemble into a fluorescent complex. In this proposal, we will use a recently developed two- component system based on the green fluorescent protein mNeonGreen2 (split-NG). By expressing one component of the split-NG pair under a tissue specific promoter while fusing the second component to a protein of interest via genomic ?knock-in?, our technique will enable tissue-specific examination of broadly expressed proteins. This technique has the potential to open new lines of inquiry in many fields of biological and biomedical research.